The sorting signal motif for targeting pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and pro-enkephalin (pro-ENK) to the regulated secretory pathway (RSP) was identified. Site directed mutagenesis studies identified an amphipathic loop at the N-terminus between Cys2 and Cys8 as the POMC sorting signal. Two pairs of acidic/hydrophobic residues (D10L11; E14L18) are exposed on the surface of the loop which are involved in binding to the sorting receptor. A similar sorting motif was identified in the N-terminus of pro-enkephalin consisting of two pairs of acidic/hydrophobic residues (D18, I19; E29,L32) exposed on the surface of a double loop stabilized by two disulfide bridges, Cys2-Cys24 and Cys6-Cys28. A RSP sorting receptor that is specific for the sorting signal of POMC, pro-insulin, pro-enkephalin and growth hormone was identified as membrane carboxypeptidese E. (CPE) Obliteration of CPE by antisense RNA in neuro2a cells and in the Cpefat/Cpefat mouse carrying a mutation in the CPE gene resulted in the missorting of POMC, pro-enkephalin and pro-insulin to the constitutive pathway, indicating that CPE functions as a sorting receptor in vivo. Our findings support a sorting signal/receptor mechanism for sorting prohormones to the regulated secretory pathway in neuro-endocrine cells. The biosynthesis and molecular basis for the specificity of a member of the novel class of aspartic protease, Yapsin1 (YAP3p) was studied. Yapsin1, a yeast pro-hormone processing enzyme was shown to be synthesized as an inactive proenzyme and activated by removal of the pro-region via a two step, autocatalytic process. The enzyme was also cleaved into two subunits, alpha and beta, which were linked by a disulfide to form a heterodimer. Pube-chase studies using wild type and the Sec 18 yeast mutant showed that pro-Yapsin1 was first cleaved into the alpha and beta subunits in the endoplasmic reticulum, followed by autocatalytic removal of the pro-region in the Golgi. Molecular modeling of Yapsin1 revealed that it has an open, highly electronegative active site pocket in the S1 subsite, favoring substrates with a basic residue in the P1 position. The model also showed that the S6, S2', and S3' subsites were electronegative and substrates with basic residues in the P6, P2' and P3' would enhance binding and the catalylic efficiency. Specificity studies using CCK13-33 analogues showed that Yapsin1 cleaved substrates with a basic residue in P1 preferentially and the catalytic efficiency was enhanced in substrates with additional basic residues in the P6, P2', P3', and P6' positions, consistent with the predictions from the model. Cloning of mammalian homologues of Yapsin1 by screening pituitary cDNA expression librabries with Yapsin1 antibodies and pepstatin inhibitable enzymatic activity have yielded several positive clones that are currently being sequenced.